Natural killer (NK) cells are key effectors of the innate immunity, which cooperate with adaptive immunity in the protection from microbial infections, viruses, fungi, and cancer. Innate immune cells have been initially considered unable to identify and eliminate microbes without pre-sensitization; however, several studies clearly demonstrated that innate immunity recognizes microbial-associated or pathogen-associated molecular patterns (PAMPs) through their pattern recognition receptors (PRRs), including Toll-like receptors (TLRs), NOD-like receptors (NLRs), C-type lectin receptors (CLRs), and RIG-I-like receptors (RLRs) ^[1][2][1,2]. Toll receptors were first described in the mid-1990s as essential molecules for embryonic patterning in drosophila that also play a role in antifungal immunity [3].

TLRs are type I transmembrane proteins with a N-terminal leucine-rich repeat (LRRs) ectodomains that mediate PAMP recognition, a transmembrane domain, and a C-terminal Toll-interleukin 1 (IL-1) receptor (TIR) domain necessary for signal transduction ^[4][6]. TIR domains are characteristic of many adaptor proteins that interact homo-typically with the TIR domains of TLRs and IL-1 receptors as the first step in the signaling cascade.

The TLR family comprises 10 members (TLR1/TLR10) in humans and 12 members in mice (TLR1-TLR9 and TLR11-TLR13), TLR1–TLR9 being conserved in both species ^[5][10]. The TLR genes are dispersed throughout the human genome: those encoding TLR1 and TLR6 map to human chromosome 4p14, TLR2 and TLR3 to 4q31.3–q35, TLR4 to 9q32–q33, TLR5 to 1q33.3–q42, TLR7 and TLR8 to Xp22, and TLR9 to 3p21.3.

Based on their cellular localizations, human TLRs can be divided in cell surface receptors (TLR1, 2, 4, 5, 6 and 10), which are primarily designated to recognize extracellular macromolecular ligands from bacteria and fungi, and endosomal (TLR3, 7, 8 and 9), recognizing cell ligands that require internalization to generate a signal as microbial DNA or RNA ^[6][11]. In particular, the TLR2-TLR1 heterodimer recognizes triacyl lipopeptides from Gram-negative bacteria and mycoplasma, whereas the TLR2-TLR6 heterodimer recognizes diacyl lipopeptides from Gram-positive bacteria and mycoplasma ^[7][8][12,13]. TLR4 responds to lipopolysaccharide, a surface structure of Gram-negative bacteria that can cause septic shock, whereas TLR5 binds the flagellin in bacterial flagella ^[9][10][14,15]. Among endosomal TLRs, TLR3 recognizes double-stranded RNA (dsRNA) produced by a number of replicating viruses ^[11][12][13][16,17,18]. By contrast, TLR7 and TLR8 recognize single-stranded RNA (ssRNA) derived from RNA of viruses, such as vesicular stomatitis virus, HIV, influenza A and some silencing RNAs ^[14][15][16][19,20,21]. Lastly, TLR9 recognizes unmethylated 2′-deoxyribo CpG DNA motifs in bacteria and viruses ^[17][5].

In addition to responding to PAMPs, TLRs respond to danger-associated molecular patterns (DAMPs), also called alarmins, and trigger inflammatory responses. Alarmins are produced as a result of cell death and injury or by tumor cells ^[18][19][22,23]. Thus, TLRs are critical sensors for immunosurveillance against tumors. The tumor microenvironment (TME) is rich in molecules potentially able to activate TLR signaling in local antigen presenting cells (APCs) to improve anti-tumor T cell responses, such as heat shock proteins, high mobility group proteins, DNA from necrotic cells, and hyaluronic acid ^[20][21][24,25]. However, besides their role in inducing anti-tumor response, tumor cells may activate negative regulatory circuits critical for normal homeostasis of the immune system through TLRs by inducing and maintaining immune tolerance to cancer. The most active negative regulators include extracellular decoy receptors (soluble TLRs), transmembrane suppressive receptors, several miRNAs, and intracellular inhibitors ^[22][26]. After ligand engagement, TLRs form homodimers or heterodimers and undergo conformational changes to recruit downstream adaptor proteins.

2. TLR Expression in NK Cells

Controversial observations have been reported on the expression of TLRs (especially of endosomal TLRs) on human NK cells, probably due to no specific detecting antibodies. However, the use of quantitative PCR primers specifically discriminating among TLR family members allowed to detect mRNA generating reliable data about the presence of each TLR in different cell types ^{[6][23][24][25]}[11,27,30,31]. Data report that TLR family members are expressed in immune cells, but also in a variety of other cells, including vascular endothelial cells, adipocytes, cardiac myocytes, and intestinal epithelial cells. The expression level of TLRs among immune cells is variable ^[26][32]. During the last two decades, the group of Alessandro and Lorenzo Moretta highlighted and repeatedly confirmed the presence of all endosomal TLRs on NK cells by different methods (mRNA expression, Western Blot analysis, and confocal microscopy) performed on purified NK cells, NK92 cell lines, or NK cell clones ^{[27][28][29][30][31]}[33,34,35,36,37]. Recently, the presence of endosomal TLRs has been directly demonstrated at the protein level and functionally through the activation of purified NK cells by specific ligands/agonists, particularly that of TLR8 ^[32][38]. NK cells express all endosomal TLRs independently of their state of activation at different levels. Despite differences among endosomal TLRs, their expression has been detected both in different donors and in NK cell clones derived from the same individual ^[33][39]. As mentioned, TLR3 is overall highly expressed, followed by TLR7 and TLR8 moderately expressed and TLR9 expressed at low or undetectable level ^[23][24][25][27,30,31].

3. Endosomal TLR Agonists and Their Activity on NK Cells

3.1. TLR3 Agonists

TLR-mediated signaling pathways in NK cells are differentially regulated by TLR ligands and agonists. dsRNA produced by several viruses is known to stimulate TLR3 both on the cell surface and intracellularly, inducing NK cell cytotoxicity and production of CXCL10, IFN-γ and other inflammatory cytokines. In particular, upon stimulation, TLR3 interacts with the adaptor protein TRIF, also known as Toll-interleukin I receptor domain containing molecule 1 (TICAM), to induce the activation of interferon regulatory factor 3 (IRF-3) and nuclear factor kB (NF-kB) transcription factors that, in turn, activate the production of inflammatory cytokines. TLR3 can be also triggered by different synthetic molecules, including polyinosinic polycytidylic [Poly (I:C)] acid and its analogs. Poly (I:C) is a synthetic analogue of dsRNA acting as an agonist, not only of TLR3, but also of retinoic acid-inducible gene RLRs, that, once triggered, in turn regulates the adaptive immune system ^{[34][35][36][37][38]}[43,44,45,46,47]. Two analogues of Poly (I:C) have been designed with the aim to reduce the toxicity related to poly (I:C) administration: Poly-IC12U and Poly-ICLC are characterized by a high molecular stability and resistance to nucleic acid hydrolysis, respectively ^[39][40][49,50]. A second synthetic molecule triggering TLR3 is RGC100, a 100 bp long dsRNA which is characterized by a high solubility caused by its chemical structure ^[41][51] and serum stability, due to the 100% CG content ^[42][52]. These characteristics play a role in reducing the potentially toxic effects that are caused by other TLR3 agonists, such as Poly (I:C) ^[43][53]. A TLR3 agonist widely used in clinical practice is ARNAX, a DNA–dsRNA hybrid compound containing dsRNA (sequence of measles virus) so that it does not induce RNAi in human transcripts. This ligand is specific for TLR3 triggering the TICAM-1 pathway only. Notably, its conjunction sites of DNA–RNA and dsRNA regions show resistance to serum nucleases ^[44][54]. Compared to Poly (I:C), ARNAX induces poor inflammatory IFN-β and cytokine production in a TLR3–TICAM-1-dependent manner, indicating that the TLR3–TICAM-1 pathway causes a not significant and localized release of cytokines under priming DCs. Endosomal TLRs expressed by NK cells are also present in other innate immune cells, therefore the triggering of certain TLRs may lead to the simultaneous activation of different cell types. In particular, NK cells share a high expression of TLR3 with DCs; this contributes to promote the bidirectional crosstalk between each other occurring in the periphery or in secondary lymphoid tissues.

3.2. TLR7/8 Agonists

TLR7 and TLR8 are two endosomal receptors existing as a heterodimer (TLR7/8). TLR7 is characterized by having two binding sites: the first is devoted to interact with small ligands and is conserved in both TLR7 and TLR8, and the second site differs from that of TLR8 and is used to bind ssRNA and enhances activation of the first site ^[45][57]. While TLR8 is triggered by ssRNA AU- and GU-rich sequences ^[46][58], TLR7 is only activated by GU-rich sequences ^[47][59]. TLR7/8 agonists include resiquimod (R848), a small-molecular-weight synthetic compound belonging to the imidazoquinoline family and other molecules specific for either TLR7 or TLR8. Among those specific for TLR7 are imiquimod, also called Aldara or R-837, whose structure is similar to an adenosine nucleoside, gardiquimod, which is similar to imiquimod, with which it shares an imidazoquinoline structure, but it has stronger properties than the latter. An additional synthetic molecule related to imiquimod is 852A, described as a more potent and selective TLR7 agonist than imiquimod ^[48][60]. Different from imiquimod are: i. loxoribine, a guanosine analogue derivatized at position N7 and C8 (7- allyl-8-oxoguanosine) that enhances NK cells activity and induces production of cytokines such as IFNs ^[49][61], ii. bropirimine (2-amino-5-bromo-6-phenyl-4-pyrimidinone), an orally administered modulator that induces production of cytokines including IFN-α ^[50][62], iii. GS-9620, a potent and selective oral TLR7 that manifested a strong antiviral activity ^[51][52][53][63,64,65] and iv. SC1, a small molecule agonist of TLR7, stimulating in particular NK cells and mediating an efficient immune response. SC1 also showed an effective anti-metastatic activity in vivo ^[54][66]. Other TLR7 specific agonists include 3M-052, 3M-011, DSR-6434, and SZU-101, all showing anti-tumor activity in human cancer models ^[55][67].

3.3. TLR9 Agonists

Plasmacytoid dendritic cells (pDCs) share with NK cells also the expression of TLR9, recognizing unmethylated 2′-deoxyribo CpG DNA motifs in bacteria and viruses ^[56][70]. Its stimulation of pDCs induces IFN-α release, further supporting the activation of TLR9-responsive NK cells. Three distinct types of synthetic oligo-deoxy-nucleotides (ODNs) containing unmethylated CpG dinucleotides have been synthetized: i. A-type ODNs, ii. B-type ODNs and iii. C-type ODNs. All ODNs activate pDCs and induce the release of inflammatory cytokines, such as TNF-α and IFN-α ^[57][71]. TLR9 recognizes also the insoluble crystal hemozoin, originated as a byproduct of detoxification after digestion of host hemoglobin by plasmodium falciparum ^[58][72]. Interestingly, the Killer immunoglobulin-like receptor 3DL2 (KIR3DL2), a NK receptor well-known for recognizing class I human leukocyte antigen (HLA), is also able to bind ODNs and to induce KIR3DL2 down-modulation from the cell surface and translocation to the endosome to deliver ODNs toTLR9 ^[59][73].

4. Endosomal TLR Agonists Stimulating NK Cells in Cancer Immunotherapy (CIT)

4.1. TLR3 Agonists in CIT

Poly (I:C) has been largely tested in vitro and in vivo to assess NK and cytotoxic T-cell (CTL) activity ^[60][75], with a consequent reduction of tumor masses in tumor bearing mice. Unfortunately, the Poly (I:C) dose necessary to obtain an adequate response provokes several side effects, such as fever, erythema, or life-threatening endotoxin-like shock, probably due to a cytokine storm induced by the reagent ^[61][97]. Poly (I:C) is not only specific for TLR3, but it can also trigger cytoplasmic receptors such as RIG-I and MDA5 ^[62][98], thus improving the inflammatory cytokine production, as IL-6, TNF-α, and IFN-β ^[35][44]. Based on these premises, it became necessary to synthetize new agonists specific for TLR3 in order to minimize the cytokine production. Among them, cM362-140 is a sODN-dsRNA conjugate able to activate the TLR3/TICAM-1 pathway in the DC endosome. cM362-140 resulted in promoting NK activation and CTL proliferation in melanoma and lymphoma mouse models, respectively. Importantly, cM362-140 triggers exclusively TLR3, provoking an anti-tumor response without causing a cytokine storm ^[63][76]. Similarly, TAARD, a synthetic disaccharide derivative of diphyllin, is able to directly trigger TLR3 on primary NK cells and activate the STAT3 pathway. In particular, TAARD increases IFN-γ production by NK cells, and presents an additive effect in combination with IL-12 or IL-15 ^[64][77]. The use of anti-programmed death receptor 1 (PD-1) and anti-cytotoxic T lymphocyte–associated antigen 4 (CTLA4) monoclonal antibodies (mAbs) is often associated with conventional chemotherapy and radiotherapy treatments to improve patients’ survival. Nevertheless, a large proportion of patients still remain unresponsive to immune checkpoint treatment (ICT), but the association with TLR agonists improves ICT efficacy ^[65][99]. In this case, responsive mesothelioma and renal murine tumors expressed higher amounts of inflammatory genes, downregulated IL-10RA gene expression and increased activation of signal transducer and activator of transcription 1 (STAT1). An example is the administration of anti-PD1 and anti-CTLA4 mAbs in non-responsive cancer animal models pretreated with IFN-γ, whose efficacy are increased by the treatment with anti-IL-10 mAb and Poly (I:C). In this context, it has also been demonstrated that TLR3 triggering induces STAT1 phosphorylation, leading to higher IFN-γ production by circulating CD335⁺, CD11b⁺ and KLRG1⁺ NK cells recruited in TME ^[66][100].

4.2. TLR 7/8 Agonists in CIT

R848 is the best known TLR7/8 agonist used for CIT. Cheadle et al. treated CD20⁺ lymphoma-bearing mice with obinutuzumab plus R848 and obtained the increase of NK cell function as activation, cytokine release, and ADCC ^[67][78]. Despite several positive results in terms of immune activation against cancer obtained with R848, its application is limited by the high toxicity and solubility, with a limited systemic employment ^[68][79]. MEDI9197 is a dual TLR 7/8 agonist linked with a lipid tail in order to reduce solubility and improve retention at the injection site. MEDI9197 increases CD25 expression on NK cells and improves their cytotoxicity ^[69][81]. Moreover, the combination of cetuximab with small synthetic compounds able to trigger both TLR7 and TLR8 in a dose dependent manner induced tumor growth inhibition and increased NK cell-mediated ADCC in a lung murine model ^[70][104]. ADCC was also improved by using the T7-MG7 compound that is made up by a small TLR7 agonist (T7) with a gastric cancer antigen (MG7) and is characterized by low toxicity and long-term effect ^[71][87].

4.3. TLR9 Agonists in CIT

In the last decade, it was reported that TLR9 ligands can directly trigger TLR9 on NK cells ^[59][73]. TLR9 agonists are mostly used in combination with inhibitory checkpoint antibodies, as anti-PD1 mAb, in non-responding cancer patients ^[72][106]. A study analyzed the activity of HP06T07I, a new ODN TLR9 agonist, to promote NCR1⁺ NK cell infiltration in a colon carcinoma mouse model ^[73][90]. NK cell recruitment in non-small-cell lung cancer (NSCLC) model was also obtained by using litenimod (Li28), a TLR9 agonist, in combination with the immunotherapeutic vaccine based on modified vaccinia virus Ankara (MVA), namely TG4010 ^[74][91]. In addition, it was demonstrated that JAK1/JAK2 mutation in melanoma patients, which provokes resistance to anti-PD1 mAb treatment, can be overcome with the treatment of SD-101 TLR9 agonist. After treatment, it was possible to detect the NK cell recruitment in the tumor site, with improved ability in controlling tumor growth ^[75][92]. MGN1703 effect was evaluated in many infectious diseases, such as HIV-1 infection. This compound acts especially on CD56^dim NK cells, in which it improves degranulation activity, IFN-γ production, NKp46 and NKG2A receptor expression ^[76][93].

5. Clinical Trials with Endosomal TLR Agonists Stimulating NK Cells

At present, 62 clinical trials (active, recruiting, completed, or terminated) on the TLR agonist employment for CIT are registered, and most of them involve the endosomal TLR agonists in combination with other treatments (chemotherapy, radiotherapy, or mAbs treatment)(https://clinicaltrials.gov). The VTX-2337 molecule (USAN: motolimod) has been used in 11 clinical trials against solid tumors, such as ovarian and squamous cell carcinoma of the head and neck (SCCHN). In particular, the studies of Chow L. and Dietsch G. N were focused on VTX-2337 in combination with cetuximab for the treatment of SCCHN patients in two phase II clinical trials (NCT01836029, NCT01334177); in this context, they observed an increased NK cell-mediated ADCC ^[77][78][88,107]. Other studies employed MGN1703 for treatment of colorectal carcinoma in combination with chemotherapy treatment. They evaluated especially the frequencies of NK and NKT cells as positive biomarkers in colorectal carcinoma patients (NCT01208194) ^[79][94]. Therapeutic advances in childhood leukemia (TACL) and lymphoma phase I consortium performed a pilot study on three patients with minimal residual disease (MRD) positive acute leukemia (NCT01743807) by using GNKG168 as a TLR9 agonist. They demonstrated that the SIGIRR, IL1RL1, CCR8, IL7R, CD8B, and CD3D genes are downregulated after GNKG168 treatment. In particular, IL7R decrease could promote CD56^bright NK cell subset suppression of graft-versus-host disease (GvHD) in acute leukemia patients ^[80][95]. Nevertheless, most of clinical trials didn’t achieve the desired therapeutic effect and provoked different adverse reactions. 

6. Novel Approaches of CIT with TLR-Activated NK Cells

Targeted drug delivery can significantly influence the efficacy of the molecule in terms of pharmacokinetics and bioavailability ^[81][111]. In the past decades, diverse drug-delivery technologies, including nano- and microparticles, co-crystals, and microneedles have been developed to maximize therapeutic efficacy and minimize unwanted side effects of therapeutics. In particular, vaccine development is often accompanied by adjuvants that promote strong T cell responses by prolonging antigen presentation by DCs and by activating NK cells ^[82][112]. Aluminum salts are the most widely applied adjuvants in human vaccines since they are safe and well tolerated ^[83][113]. The micrometer-sized aluminum aggregates were transformed into nano-sized vaccine carriers by shielding their positive charges with a polyethylene glycol (PEG)-containing polymer ^[84][114]. Internalization of these molecules is highly dependent on scavenger receptor A-mediated endocytosis ^[84][114]. Polymeric nanoparticles (NPs) and liposomes have been investigated to encapsulate payloads, including drugs, proteins, vectors, and nucleic acids ^[85][115]. Moreover, NPs tend to accumulate in tumors and not in normal tissue as a consequence of leaky tumor vasculature and damaged lymphatic drainage ^[86][116]. In the last few years, nanomaterials have been designed to boost NK cell CIT. In particular, most NPs were developed to modify the immunosuppressive TME ^[87][117] so as to improve the recruitment of NK cells in tumor sites ^[88][89][101,118] and to restore NK cells’ function by increasing their proliferation, cytotoxicity and cytokine production ^[90][119]. Many studies assessed NP loaded with TLR agonists, alone or in combination with other molecules ^[91][120]. For example, PLGA-NP co-loaded with indocyanine green (ICG) fluorescent dye and R848 (PLGA-ICG-R848) was proposed for treatment of prostate cancer, where it induced an increase of cytotoxic activity of NK cells ^[92][80]. Kim, H. et al. used a pH-responsive polymeric NP wherein they encapsulated a TLR7/8 agonist ^[93][121]. The treatment with this compound prolonged NK cell activation and improved in vivo cytotoxicity much more than using a soluble agonist. In addition, TLR7/8 agonist-loaded NPs potentiated NK cell mediated ADCC in combination with cetuximab in melanoma model ^[94][122].